CN112010936A - 乙基grpak修饰的双咔啉并哌嗪二酮,其制备,活性和应用 - Google Patents
乙基grpak修饰的双咔啉并哌嗪二酮,其制备,活性和应用 Download PDFInfo
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Abstract
Description
技术领域
本发明涉及(2’S,5’S)-四氢吡嗪[1’,2’:1,6]并双[1S,1R-(1-乙基-Gly-Arg-Pro-Ala-Lys)-2,3,4,9-四氢-1H-吡啶[3,4-b]并吲哚]-1’,4’-二酮,涉及它的制备方法,涉及它的溶血栓活性,涉及它的抗血栓活性以及涉及它对缺血性中风发作24小时仍然有效的特征。因而本发明涉及它在制备溶血栓药物中的应用,涉及它在制备抗血栓药物中的应用,涉及它在制备缺血性中风发作24小时仍然有效的药物中的应用以及涉及它在制备具有溶血栓,抗血栓及对缺血性中风发作24小时仍然有效的三重作用的药物中的应用。本发明属于生物医药领域。
技术背景
缺血性中风是常见的危害严重的脑血管疾病。缺血性中风以发病率高,致残率高,复发率高和死亡率高为特点,是人类最严重的致死性疾病之一。目前,rtPA是临床公认的治疗缺血性中风的唯一有效药物。然而,rtPA治疗缺血性中风存在两个难以逾越的难题。第一个难题是rtPA对缺血性中风发作4小时以上的患者没有疗效。第二个难题是连续使用rtPA可引起脑,胸腔及腹腔出血。发明对中风发作4小时以上尤其对发作24小时患者有效又无出血副作用的药物是脑血管药物研究的热点与前沿。经过数年探索,发明人发现在双咔啉并哌嗪二酮引入乙基-Gly-Arg-Pro-Ala-Lys生成的下式的(2’S,5’S)-四氢吡嗪[1’,2’:1,6]并双[1S,1R-(1-乙基-Gly-Arg-Pro-Ala-Lys)-2,3,4,9-四氢-1H-吡啶[3,4-b]并吲哚]-1’,4’-二酮不仅有溶血栓活性,有抗血栓活性,对缺血性中风发作24小时仍有疗效,而且没有出血副作用。根据这些发现,发明人提出了本发明。
发明内容
本发明的第一个内容是提供下式的(2’S,5’S)-四氢吡嗪[1’,2’:1,6]并双[1S,1R-(1-乙基-Gly-Arg-Pro-Ala-Lys)-2,3,4,9-四氢-1H-吡啶[3,4-b]并吲哚]-1’,4’-二酮。
本发明的第二个内容是提供(2’S,5’S)-四氢吡嗪[1’,2’:1,6]并双[1S,1R-(1-乙基-Gly-Arg-Pro-Ala-Lys)-2,3,4,9-四氢-1H-吡啶[3,4-b]并吲哚]-1’,4’-二酮的制备方法,该方法包括以下7个步骤:
(1)制备(3S)-1-(2,2-二甲氧乙-1-基)-2,3,4,9-四氢-β-咔啉-3-羧酸苄酯;
(2)制备(3S)-1-(2,2-二甲氧乙-1-基)-2,3,4,9-四氢-β-咔啉-3-羧酸;
(3)制备(2’S,5’S)-四氢吡嗪[1’,2’:1,6]并双[1S,1R-(1-二甲氧乙-1-基)-2,3,4,9-四氢-1H-吡啶[3,4-b]并吲哚]-1’,4’-二酮;
(4)制备(2’S,5’S)-四氢吡嗪[1’,2’:1,6]并双[1S,1R-(1-羰甲基)-2,3,4,9-四氢-1H-吡啶[3,4-b]并吲哚]-1’,4’-二酮;
(5)采用N,N’-二环己基碳二亚胺为缩合剂,1-羟基苯并***为催化剂的液相法合成Gly-Arg(NO2)-Pro-Ala-Lys(Cbz)-OBzl;
(6)采用氨化还原法将(2’S,5’S)-四氢吡嗪[1’,2’:1,6]并双[1S,1R-(1-羰甲基)-2,3,4,9-四氢-1H-吡啶[3,4-b]并吲哚]-1’,4’-二酮与Gly-Arg(NO2)-Pro-Ala-Lys(Cbz)-OBzl反应制备(2’S,5’S)-四氢吡嗪[1’,2’:1,6]并双[1S,1R-(1-乙基-Gly-Arg(NO2)-Pro-Ala-Lys(Cbz)-OBzl)-2,3,4,9-四氢-1H-吡啶[3,4-b]并吲哚]-1’,4’-二酮;
(7)制备(2’S,5’S)-四氢吡嗪[1’,2’:1,6]并双[1S,1R-(1-乙基-Gly-Arg-Pro-Ala-Lys)-2,3,4,9-四氢-1H-吡啶[3,4-b]并吲哚]-1’,4’-二酮。
本发明的第三个内容是评价(2’S,5’S)-四氢吡嗪[1’,2’:1,6]并双[1S,1R-(1-乙基-Gly-Arg-Pro-Ala-Lys)-2,3,4,9-四氢-1H-吡啶[3,4-b]并吲哚]-1’,4’-二酮的溶血栓活性。
本发明的第四个内容是评价(2’S,5’S)-四氢吡嗪[1’,2’:1,6]并双[1S,1R-(1-乙基-Gly-Arg-Pro-Ala-Lys)-2,3,4,9-四氢-1H-吡啶[3,4-b]并吲哚]-1’,4’-二酮的抗血栓活性。
本发明的第五个内容是评价(2’S,5’S)-四氢吡嗪[1’,2’:1,6]并双[1S,1R-(1-乙基-Gly-Arg-Pro-Ala-Lys)-2,3,4,9-四氢-1H-吡啶[3,4-b]并吲哚]-1’,4’-二酮对缺血性中风发作24小时的大鼠的疗效。
附图说明
图1(2’S,5’S)-四氢吡嗪[1’,2’:1,6]并双[1S,1R-(1-乙基-Gly-Arg-Pro-Ala-Lys)-2,3,4,9-四氢-1H-吡啶[3,4-b]并吲哚]-1’,4’-二酮的合成路线。i)三氟乙酸,二氯甲烷,40℃;ii)NaOH水溶液(2M),丙酮,0℃;iii)2-(7-偶氮苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯,N-甲基吗啉,N,N-二甲基甲酰胺;iv)冰醋酸,水;v)N,N’-二环己基碳二亚胺,1-羟基苯并***,N-甲基吗啉,四氢呋喃;vi)NaOH水溶液(2M),甲醇,0℃;vii)氯化氢的乙酸乙酯溶液(4M),0℃;viii)N-甲基吗啉,氰基硼氢化钠,无水硫酸镁,二氯甲烷,甲醇;ix)钯碳(Pd/C),H2,甲醇。
具体实施方式
为了进一步阐述本发明,下面给出一系列实施例。这些实施例完全是例证性的,它们仅用来对本发明进行具体描述,不应当理解为对本发明的限制。
实施例1制备(3S)-1-(2,2-二甲氧乙-1-基)-2,3,4,9-四氢-β-咔啉-3-羧酸苄酯(1)
于0℃与搅拌下将向150mL二氯甲烷中加入5mL 1,1,3,3-四甲氧基丙烷及3.5mL三氟乙酸,反应40分钟后,加入5g(17.00mmol)Trp-OBzl,于40℃进行反应。反应4小时后,于0℃与搅拌下用浓氨水将反应液pH值调节至7,减压浓缩,残留物用100mL二氯甲烷溶解,依次用饱和碳酸氢钠水溶液洗3次,饱和氯化钠水溶液洗3次,二氯甲烷相用无水硫酸钠干燥12小时,过滤,滤液减压浓缩至干,得到黄色油状物,经硅胶柱层析纯化,得到5.50g(82%)标题化合物,为黄色油状物。ESI-MS(m/e):395[M+H]+。
实施例2制备(3S)-1-(2,2-二甲氧乙-1-基)-2,3,4,9-四氢-β-咔啉-3-羧酸(2)
将5.50g(13.96mmol)(3S)-1-(2,2-二甲氧基乙-1-基)-2,3,4,9-四氢-β-咔啉-3-羧酸苄酯(1)溶于50mL丙酮中,于0℃与搅拌下向其中加入NaOH水溶液(2M),调节溶液pH值为12,反应3小时,保持反应液pH值为12。之后,用饱和硫酸氢钾水溶液调节pH值至中性,减压浓缩。残留物中加入50mL丙酮,过滤除去不溶固体,滤液减压浓缩至干,得到3.63g(86%)标题化合物,为黄色黏状物。ESI-MS(m/e):303[M-H]-。
实施例3制备(2’S,5’S)-四氢吡嗪[1’,2’:1,6]并双[1S,1R-(1-二甲氧乙-1-基)-2,3,4,9-四氢-1H-吡啶[3,4-b]并吲哚]-1’,4’-二酮(3)
将3.63g(11.94mmol)(3S)-1-(2,2-二甲氧乙-1-基)-2,3,4,9-四氢-β-咔啉-3-羧酸(2)溶于20mL无水N,N-二甲基甲酰胺中,之后加5.44g(14.32mmol)2-(7-偶氮苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯,溶液于0℃与搅拌下用N-甲基吗啉调节溶液pH值至8,室温搅拌16小时后,向反应液中加150mL蒸馏水,之后用二氯甲烷萃取(50mL×3),合并二氯甲烷相,再用饱和氯化钠水溶液洗(50mL×3),二氯甲烷相用无水硫酸钠干燥12小时,过滤,滤液减压浓缩至干,得到的黄色油状物经硅胶柱层析纯化,得到0.73g(21%)标题化合物,为淡黄色固体。ESI-MS(m/e):573[M+H]+。
实施例4制备(2’S,5’S)-四氢吡嗪[1’,2’:1,6]并双[1S,1R-(1-羰甲基)-2,3,4,9-四氢-1H-吡啶[3,4-b]并吲哚]-1’,4’-二酮(4)
将0.30g(0.52mmol)(2’S,5’S)-四氢吡嗪[1’,2’:1,6]并双{1S,1R-(1-二甲氧乙-1-基)-2,3,4,9-四氢-1H-吡啶[3,4-b]并吲哚}-1’,4’-二酮(3)溶于20mL冰醋酸中,加入2mL蒸馏水,室温搅拌反应48小时后,过滤,滤饼用蒸馏水洗去残留的冰醋酸,晾干,得0.22g(87%)标题化合物,为无色固体。ESI-MS(m/e):479[M-H]-。
实施例5制备Boc-Pro-Ala-OBzl
将5.00g(23.25mmol)Boc-Pro溶于20mL无水四氢呋喃中,于0℃与搅拌下加入2.86g(21.18mmol)1-羟基苯并***,5.23g(25.39mmol)N,N’-二环己基碳二亚胺,于0℃与搅拌下搅拌30分钟。之后向其中加入3.79g(21.17mmol)Ala-OBzl,用N-甲基吗啉调节pH值至8,室温搅拌16小时。过滤除去不溶固体,滤液减压浓缩,残留物用100mL乙酸乙酯溶解,过滤除去不溶固体,滤液依次分别用饱和碳酸氢钠水溶液洗(30mL×3),饱和氯化钠水溶液洗(30mL×3),5%硫酸氢钾水溶液洗(30mL×3),饱和氯化钠水溶液洗(30mL×3),饱和碳酸氢钠水溶液洗(30mL×3)及饱和氯化钠水溶液洗(30mL×3),乙酸乙酯相用无水硫酸钠干燥12小时,过滤,滤液减压浓缩至干,得6.93g(87%)标题化合物,为无色固体。ESI-MS(m/e):377[M+H]+。
实施例6制备Boc-Pro-Ala
将6.93g(18.43mmol)Boc-Pro-Ala-OBzl溶于30mL甲醇中,于0℃与搅拌下向其中加入NaOH水溶液(2M),调节pH值为12,搅拌3小时,保持反应液pH值为12。之后用饱和硫酸氢钾水溶液调节pH值至中性,减压浓缩。残留物加入100mL蒸馏水,再用饱和硫酸氢钾水溶液调节pH值至2,用乙酸乙酯萃取(30mL×3),合并乙酸乙酯相,用饱和氯化钠水溶液洗(30mL×3),将乙酸乙酯相用无水硫酸钠干燥12小时,过滤,滤液减压浓缩至干,得到4.80g(91%)标题化合物,为无色油状物。ESI-MS(m/e):285[M-H]-。
实施例7制备Boc-Pro-Ala-Lys(Cbz)-OBzl
用实施例5的方法从4.80g(16.78mmol)Boc-Pro-Ala与5.64g(15.24mmol)Lys(Cbz)-OBzl得到7.65g(79%)标题化合物,为无色固体。ESI-MS(m/e):639[M+H]+。
实施例8制备Pro-Ala-Lys(Cbz)-OBzl
于0℃和搅拌下将5.00g(7.84mmol)Boc-Pro-Ala-Lys(Cbz)-OBzl用50mL氯化氢的乙酸乙酯溶液(4M)溶解,之后继续搅拌4小时。将反应液减压浓缩,残留物用50mL无水乙酸乙酯溶解,再减压浓缩。该操作反复3次。得到的无色固体用50mL无水***充分悬浮,洗去游离氯化氢,得到4.13g(98%)标题化合物,为无色固体,直接用于下步反应。
实施例9制备Boc-Arg(NO2)-Pro-Ala-Lys(Cbz)-OBzl
用实施例5的方法从2.69g(8.43mmol)Boc-Arg(NO2)及4.13g(7.68mmol)Pro-Ala-Lys(Cbz)-OBzl得到4.17g(65%)标题化合物,为无色固体。ESI-MS(m/e):840[M+H]+。
实施例10制备Arg(NO2)-Pro-Ala-Lys(Cbz)-OBzl
用实施例8的方法从2g(2.38mmol)Boc-Arg(NO2)-Pro-Ala-Lys(Cbz)-OBzl得到1.73g(98%)标题化合物,为无色固体,直接用于下步反应。
实施例11制备Boc-Gly-Arg(NO2)-Pro-Ala-Lys(Cbz)-OBzl
用实施例5的方法从0.45g(2.57mmol)Boc-Gly及1.73g(2.34mmol)Arg(NO2)-Pro-Ala-Lys(Cbz)-OBzl得到0.96g(46%)标题化合物,为无色固体。ESI-MS(m/e):897[M+H]+。
实施例12制备Gly-Arg(NO2)-Pro-Ala-Lys(Cbz)-OBzl
用实施例8的方法从0.57g(0.64mmol)Boc-Gly-Arg(NO2)-Pro-Ala-Lys(Cbz)-OBzl得到0.48g(94%)标题化合物,为无色固体,直接用于下步反应。
实施例13制备(2’S,5’S)-四氢吡嗪[1’,2’:1,6]并双[1S,1R-(1-乙基-Gly-Arg(NO2)-Pro-Ala-Lys(Cbz)-OBzl)-2,3,4,9-四氢-1H-吡啶[3,4-b]并吲哚]-1’,4’-二酮(5)
将0.48g(0.60mmol)Gly-Arg(NO2)-Pro-Ala-Lys(Cbz)-OBzl溶于7mL二氯甲烷中,用N-甲基吗啉将pH调至8,得到反应液A。将0.10g(0.21mmol)(2’S,5’S)-四氢吡嗪[1’,2’:1,6]并双[1S,1R-(1-羰甲基)-2,3,4,9-四氢-1H-吡啶[3,4-b]并吲哚]-1’,4’-二酮(4)悬浮于5mL二氯甲烷,用N-甲基吗啉将pH调至8,得到反应液B。先将反应液B加入到反应液A中,然后依次加50mg无水硫酸镁和3mL甲醇。得到的反应混合物室温搅拌,并每隔30分钟加一次氰基硼氢化钠,共加4次,每次加16mg(0.26mmol)总共加64mg(1.03mmol)。反应混合物室温搅拌16小时。之后,反应混合物减压浓缩,残留物溶解于20mL二氯甲烷及5mL甲醇中,滤去不溶物,滤液减压浓缩,得到的淡黄色黏状固体经硅胶柱层析纯化,得到0.08g(19%)标题化合物,为无色固体。ESI-MS(m/e):2041[M+H]+.1H NMR(300MHz,DMSO-d6):δ/ppm=11.07~11.13(m,2H),7.82~8.50(m,12H),7.48(m,2H),7.34(m,22H),7.23(m,2H),7.08(m,2H),6.99(m,2H),5.80(m,1H),5.30(m,1H),5.10(m,4H),4.99(m,4H),4.56(m,1H),4.46(m,2H),4.22~4.36(m,7H),3.54~3.63(m,4H),3.08~3.18(m,8H),2.95(m,6H),1.95~2.27(m,8H),1.52~1.80(m,14H),1.24~1.38(m,8H),1.09~1.20(m,10H).13C NMR(125MHz,DMSO-d6):δ/ppm=173.0,172.3,171.5,171.2,170.2,170.1,168.9,165.0,159.7,156.6,137.7,136.6,136.5,136.4,133.5,128.9,128.8,128.5,128.3,128.2,126.5,126.3,121.7,121.5,119.2,118.3,118.2,111.7,107.2,105.3,66.4,65.6,59.6,57.6,53.4,52.5,52.3,52.0,50.0,48.3,48.0,47.3,47.2,46.2,45.0,40.8,34.1,32.0,30.9,29.4,29.2,25.5,24.9,24.8,24.5,23.0,22.3,18.7,18.5。
实施例14制备(2’S,5’S)-四氢吡嗪[1’,2’:1,6]并双[1S,1R-(1-乙基-Gly-Arg-Pro-Ala-Lys)-2,3,4,9-四氢-1H-吡啶[3,4-b]并吲哚]-1’,4’-二酮(6)
将0.08g(0.04mmol)(2’S,5’S)-四氢吡嗪[1’,2’:1,6]并双[1S,1R-(1-乙基-Gly-Arg(NO2)-Pro-Ala-Lys(Cbz)-OBzl)-2,3,4,9-四氢-1H-吡啶[3,4-b]并吲哚]-1’,4’-二酮(5)溶于5mL二氯甲烷及15mL甲醇的混合溶剂中。然后依次向内加20mg钯碳(Pd/C)和2滴甲酸。反应混合物室温通氢气并搅拌16小时。之后,滤去Pd/C。滤液减压浓缩,残留物用5mL二氯甲烷及15mL甲醇溶解。往得到的溶液中再加另外20mg Pd/C和2滴甲酸,然后室温通氢气并搅拌16小时。之后,滤去Pd/C,滤液减压浓缩,得到的无色固体经Sephadex纯化,得到0.03g(51%)标题化合物,为无色固体。Mp 237~238℃;(c=0.1,H2O);ESI-MS(m/e):1503[M+H]+.1H NMR(300MHz,MeOD):δ/ppm=7.50(m,2H),7.39(m,2H),7.18(m,2H),7.07(m,2H),5.75~6.07(m,1H),5.53(m,1H),4.70(m,2H),4.44~4.57(m,3H),4.32(m,5H),3.73~3.96(m,6H),3.49~3.54(m,2H),3.08~3.25(m,6H),2.80~3.00(m,8H),2.03~2.30(m,8H),1.91(m,6H),1.70(m,14H),1.42~1.48(m,12H).13C NMR(125MHz,MeOD):δ/ppm=176.0,174.1,173.2,173.1,171.0,170.7,165.5,165.0,158.6,156.6,136.4,130.6,130.2,128.4,125.6,125.3,122.4,119.7,119.6,118.0,117.9,111.6,111.5,107.9,105.6,64.7,60.2,60.1,57.4,53.6,53.2,53.1,52.3,51.3,51.0,50.9,49.4,44.6,42.9,40.5,39.1,30.5,29.9,29.2,27.6,27.4,26.2,24.5,24.3,24.0,23.8,22.0,21.9,16.6,16.5.IR(cm-1):3150.89,3047.35,2959.41,2926.02,2851.06,1729.44,1633.40,1539.81,1447.15,1405.48,1334.20,1260.16,1200.19,1154.92,744.33,672.70,607.13。
实验例1评价化合物6的溶血栓活性
将雄性SD大鼠(200±20g)随机分组,每组8只,静息饲养一天。将大鼠用20%乌拉坦的生理盐水溶液腹腔注射麻醉。麻醉大鼠仰卧位固定,分离右颈总动脉,于近心端夹动脉夹,近心端和远心端分别穿入手术线,将远心端的手术线于皮毛用止血钳夹紧,在远心端插管,松开动脉夹,放出约1mL动脉血,装在1mL子弹头中。往垂直固定的橡胶管(长15mm,内径2.5mm,外径5.0mm,管底用胶塞密封,para膜封紧)中注入0.1mL大鼠动脉血液,往管内迅速***一支不锈钢质料的血栓固定螺栓。该血栓固定螺栓用直径为0.2mm的不锈钢丝绕成,螺旋部分长10mm,含15个螺圈,螺圈的直径为1.0mm,托柄与螺旋相连,长7.0mm,呈问号型。血液凝固40分钟后,从橡胶管中小心取出被血栓包裹的血栓固定螺栓,精确称血栓固定螺栓的起始重量。
旁路插管由三段构成。中段是长60mm内径3.5mm的聚乙烯胶管。两侧段均为长100mm,内径1.0mm,外径2.0mm的聚乙烯管。聚乙烯管的一端拉成长10mm外径1.0mm的尖(用于***大鼠颈动脉及静脉),聚乙烯管的另一端的外部套一段长7mm外径3.5mm的聚乙烯管(用于连接中段聚乙烯管)。3段管的内壁均需硅烷化(1%的硅油***溶液)。将精确称重的血栓包裹的血栓固定螺栓置于中段聚乙烯管内。
继续分离麻醉大鼠的气管,并做气管插管。分离大鼠的左颈外静脉,近心端和远心端分别穿入手术线,在暴露的左颈外静脉上小心地剪一斜口。将制好的旁路管道的尖管由斜口***左颈外静脉开口的近心端,并远离旁路管中段内血栓固定螺旋的托柄。用注射器通过另一端的尖管推入准确量的肝素钠的生理盐水溶液(50IU/kg),此时注射器不要撤离聚乙烯管。用止血钳夹住注射器与聚乙烯管之间的软管。在右颈总动脉的近心端用动脉夹止血,在离动脉夹不远处将右颈总动脉小心地剪一斜口。从聚乙烯管的尖部拔出注射器,将聚乙烯管的尖部***动脉斜口的近心端。旁路管道的两端都用4号手术缝线将动静脉固定。
将含生理盐水(3mL/kg)或尿激酶的生理盐水溶液(20000IU/kg)或化合物6的生理盐水溶液(100nmol/kg)的头皮针刺入旁路管中段的远离血栓固定螺旋的近静脉处,打开动脉夹,使血流通过旁路管道从动脉流向静脉。将头皮针中的生理盐水(空白对照),尿激酶(阳性对照)或化合物6缓慢注入旁路管中段,使之通过血液循环按静脉-心脏-动脉的顺序作用到血栓上。从开始注射时计时,1小时后从旁路管中段取出血栓固定螺栓,精确称血栓固定螺栓的终末重量。血栓固定螺栓的起始重量与终末重量的差即为血栓减重。计算每只大鼠的血栓减重,统计各组大鼠的血栓减重并做t检验。实验数据见表1。可以看到,在100nmol/kg剂量下化合物6治疗的大鼠的血栓减重(30.42±3.33mg)显著大于生理盐水治疗的大鼠的血栓减重(23.18±1.40mg,P<0.01)。此外,在100nmol/kg剂量下化合物6治疗的大鼠的血栓减重与20000IU/kg剂量尿激酶治疗的大鼠的血栓减重(27.61±3.47mg,P>0.05)无显著性差异。化合物6的优秀溶血栓活性说明本发明有突出的技术效果。
表1化合物6的溶血栓活性
a)与生理盐水比P<0.01;b)与生理盐水比P<0.01,与尿激酶比P>0.05;n=8.
实验例2评价化合物6的抗血栓活性
本实验使用与实验例1相同的旁路插管。将雄性SD大鼠(200±20g),随机分组,每组10只,饲养1天,停止喂食过夜。灌胃给予化合物6的生理盐水溶液(剂量100nmol/kg)或阿司匹林的生理盐水溶液(剂量167μmol/kg)或生理盐水(剂量3mL/kg)。给药30分钟后,将大鼠用20%乌拉坦的生理盐水溶液腹腔注射麻醉。麻醉后将大鼠固定于大鼠固定板上,分离大鼠的右颈动脉和左颈静脉,将准确称重的丝线置于旁路插管,管的一端***左静脉,另一端管***右侧动脉并注射0.2mL肝素钠抗凝。使得血流从右侧动脉流经旁路插管进入左侧静脉,15分钟之后取出附有血栓的丝线称量,计算血液循环前后丝线的重量差。该差值即为血栓重,代表抗血栓活性。统计各组大鼠的血栓重,作t检验。实验数据见表2。可以看出,在100nmol/kg口服剂量下化合物6治疗的大鼠的栓重(17.88±3.19mg)显著小于生理盐水治疗的大鼠的栓重(26.55±4.61mg,P<0.01)。此外,在100nmol/kg口服剂量下化合物6治疗的大鼠的栓重与167μmol/kg口服剂量下阿司匹林治疗的大鼠的栓重(17.08±2.80mg,P>0.05)相比无显著性差异。也就是说,即使剂量低1670倍化合物6的抗动脉血栓活性仍然与阿司匹林相当。化合物6的优秀抗血栓活性说明本发明有突出的技术效果。
表2化合物6的抗血栓活性
a)与生理盐水比P<0.01;b)与生理盐水比P<0.01,与阿司匹林比P>0.05;n=10.
实验例3评价化合物6对缺血性中风24小时大鼠的治疗作用
雄性SD大鼠(280±20g)用10%水合氯醛(4mL/kg)腹腔注射麻醉。在大鼠的颈部正中部竖直开约2cm长切口,沿胸锁乳突肌内侧缘分离出右颈总动脉,颈外动脉及颈内动脉。用无创动脉夹分别夹闭颈内动脉开口处和颈总动脉近心端,结扎颈外动脉的远心端,在颈外动脉剪一小口,松开颈总动脉近心端的动脉夹,取10μL血入1mL离心管。之后,再用无创动脉夹夹闭颈总动脉的近心端。将离心管中的血常温放置30分钟使凝固,然后-20℃过夜使血块凝结实。取出血液凝块,加入1mL生理盐水,用钢铲把血液凝块捣成大小均一的细小血栓块,制备细小血栓的悬液并转移至1mL注射器内。松开大鼠颈总动脉近心端的动脉夹,将1mL血栓混悬液缓慢从大鼠颈外动脉向近心端经过颈内动脉注入大鼠的大脑,然后结扎颈外动脉近心端,打开颈内动脉和颈总动脉处的动脉夹,恢复血流。等待苏醒。大鼠苏醒24小时后按Zealonga方法评定神经功能缺损程度。0分表示无任何神经功能缺失体征,1分表示未损伤侧前肢不能伸展,2分表示向未损伤侧行走,3分表示向未损伤侧转圈成追尾状行走,4分表示意识障碍无自主行走,5分表示死亡。按照得分将大鼠均匀分组。各组大鼠每天经尾静脉注射1次生理盐水(3mL/kg/d)或rtPA(3mg/kg/d)或化合物6(10nmol/kg/d)。连续注射3天,每天评分。根据评分计算各组大鼠死亡率=死亡只数/总只数×100%;显效率=最后一天评分低于给药前评分但不为0的只数/总只数×100%;治愈率=最后一天评分为0的只数/总只数×100%。数据见表3,表4及表5。结果表明,化合物6治疗的大鼠的死亡率低于生理盐水治疗的大鼠的死亡率而治愈率远远高于生理盐水治疗的治愈率。此外,化合物6治疗大鼠的死亡率低于rtPA治疗大鼠的死亡率而治愈率远远高于rtPA的治愈率。rtPA治疗引起大鼠全身出血,化合物6没有出血副作用。可见,本发明有突出的技术效果。
表3生理盐水对缺血性中风发作24小时大鼠神经生物学评分的影响
表4 rtPA对缺血性中风发作24小时大鼠神经生物学评分的影响
表5化合物6对缺血性中风发作24小时大鼠神经生物学评分的影响
Claims (6)
2.权利要求1的(2’S,5’S)-四氢吡嗪[1’,2’:1,6]并双[1S,1R-(1-乙基-Gly-Arg-Pro-Ala-Lys)-2,3,4,9-四氢-1H-吡啶[3,4-b]并吲哚]-1’,4’-二酮的制备方法,该方法包括以下7个步骤:
(1)制备(3S)-1-(2,2-二甲氧乙-1-基)-2,3,4,9-四氢-β-咔啉-3-羧酸苄酯;
(2)制备(3S)-1-(2,2-二甲氧乙-1-基)-2,3,4,9-四氢-β-咔啉-3-羧酸;
(3)制备(2’S,5’S)-四氢吡嗪[1’,2’:1,6]并双[1S,1R-(1-二甲氧乙-1-基)-2,3,4,9-四氢-1H-吡啶[3,4-b]并吲哚]-1’,4’-二酮;
(4)制备(2’S,5’S)-四氢吡嗪[1’,2’:1,6]并双[1S,1R-(1-羰甲基)-2,3,4,9-四氢-1H-吡啶[3,4-b]并吲哚]-1’,4’-二酮;
(5)采用N,N’-二环己基碳二亚胺为缩合剂,1-羟基苯并***为催化剂的液相法合成Gly-Arg(NO2)-Pro-Ala-Lys(Cbz)-OBzl;
(6)采用氨化还原法将(2’S,5’S)-四氢吡嗪[1’,2’:1,6]并双[1S,1R-(1-羰甲基)-2,3,4,9-四氢-1H-吡啶[3,4-b]并吲哚]-1’,4’-二酮与Gly-Arg(NO2)-Pro-Ala-Lys(Cbz)-OBzl反应制备(2’S,5’S)-四氢吡嗪[1’,2’:1,6]并双[1S,1R-(1-乙基-Gly-Arg(NO2)-Pro-Ala-Lys(Cbz)-OBzl)-2,3,4,9-四氢-1H-吡啶[3,4-b]并吲哚]-1’,4’-二酮;
(7)制备(2’S,5’S)-四氢吡嗪[1’,2’:1,6]并双[1S,1R-(1-乙基-Gly-Arg-Pro-Ala-Lys)-2,3,4,9-四氢-1H-吡啶[3,4-b]并吲哚]-1’,4’-二酮。
3.权利要求1的(2’S,5’S)-四氢吡嗪[1’,2’:1,6]并双[1S,1R-(1-乙基-Gly-Arg-Pro-Ala-Lys)-2,3,4,9-四氢-1H-吡啶[3,4-b]并吲哚]-1’,4’-二酮在制备溶血栓药物中的应用。
4.权利要求1的(2’S,5’S)-四氢吡嗪[1’,2’:1,6]并双[1S,1R-(1-乙基-Gly-Arg-Pro-Ala-Lys)-2,3,4,9-四氢-1H-吡啶[3,4-b]并吲哚]-1’,4’-二酮在制备抗血栓药物中的应用。
5.权利要求1的(2’S,5’S)-四氢吡嗪[1’,2’:1,6]并双[1S,1R-(1-乙基-Gly-Arg-Pro-Ala-Lys)-2,3,4,9-四氢-1H-吡啶[3,4-b]并吲哚]-1’,4’-二酮在制备对缺血性中风发作24小时仍然有效的药物中的应用。
6.权利要求1的(2’S,5’S)-四氢吡嗪[1’,2’:1,6]并双[1S,1R-(1-乙基-Gly-Arg-Pro-Ala-Lys)-2,3,4,9-四氢-1H-吡啶[3,4-b]并吲哚]-1’,4’-二酮在制备具有溶血栓,抗血栓及对缺血性中风发作24小时仍然有效的三重作用的药物中的应用。
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